Nontypable Haemophilus influenzae is a common cause of localized respiratory tract disease, especially otitis media, sinusitis, bronchitis, and pneumonia. In addition, this organism causes serious systemic disease, such as meningitis, endocarditis, and septicemia. The initial step in the pathogenesis of nontypable H. influenzae disease involves colonization of the upper respiratory mucosa. In studies with cultured epithelial cells, we have identified an H. influenzae serine protease called Hap, which facilitates intimate interaction with the epithelial surface and also promotes bacterial aggregation and microcolony formation. Based on our in vitro results, we speculate that Hap plays an important role in the process of colonization. Hap shares significant homology with the H. influenzae and Neisseria gonorrhoeae IgA1 proteases and belongs to the growing family of autotransporter proteins elaborated by pathogenic gram-negative bacteria. Like other members of this family, Hap is synthesized as a precursor protein with 3 functional domains, including an N-terminal signal sequence, an internal protease domain with adhesive activity (Hap-s), and a C-terminal outer membrane domain with translocator activity (Hap-beta). Ultimately, Hap undergoes autoproteoiytic cleavage, with extracellular release of Hap-s. In recent work, we demonstrated that Hap-mediated adherence and microcolony formation are potentiated by a host protein called secretory leukocyte protease inhibitor (SLPI). This protein is present in respiratory secretions and inhibits Hap autoproteolysis, resulting in accumulation of surface-associated Hap-s. In the present proposal, we will focus on Hap-mediated adherence and microcolony formation. In Aim 1, we will solve the crystal structure of Hap-s and define the interactive surfaces involved in adherence and microcolony formation. In Aim 2, we will examine the ability of microcolonies to resist lactoferrin protease activity, to evade macrophage phagocytosis, and to enhance persistence in the chinchilla otitis media model. In Aim 3, we will characterize the relationship between respiratory viral infection and Hap-mediated adherence and microcolony formation, concentrating on the role of SLPI. From a practical perspective, the proposed studies may facilitate efforts to develop novel strategies for the treatment and prevention of H. influenzae disease. Perhaps more importantly, they may provide general insights into host-microbe relationships and expand our understanding of microbial biofilms.